Visceral pain is a debilitating and poorly understood condition that is a common cause for seeking medical attention. In comparison to somatic pain, much less is known about the visceral pain pathways. Progress in this area has been slow because visceral pain is more difficult to measure, the majority of visceral afferent nerves are involved not in pain but in autonomic regulation, and their genetic identities are often unknown. Pain can be evoked from some organs (e.g. esophagus, stomach, ureter, colon) but not from others (e.g. liver, spleen, lung), despite the fact that all visceral organs are innervated by biochemically similar afferent nerves. Our central hypothesis is that nociceptive pathways from pain-evoking organs are exclusively connected to central pain pathways compared to nociceptive pathways from other organs. Thus, a thorough comparison of afferent pathways from the organs which produce pain vs. those which do not produce pain will provide the identity the afferent visceral subsets and spinal pathways which specifically mediate visceral pain. However, such task requires previously unavailable strategies to analyze the responsiveness of peripheral nerve terminals of genetically-defined visceral afferent subsets, and to reliably trace their central connections. We have developed a battery of state-of-the-art techniques that allow for the mapping of individual afferent subsets defined by the coincidence of two genes from two organs simultaneously. Our approach will trace either the DRG afferents themselves, or the central pathways (connectomes) downstream of these specific subsets. Combined with functional assessment of the nociceptive status of DRG afferent subsets in situ by using GCaMP6 imaging, these approaches will for the first time provide knowledge of the central pathways activated by unique genetically-defined nociceptive subsets. Our collaborative team is comprised of Drs. Taylor-Clark and Kollarik, experts in visceral nociceptive afferents; Drs. Teng and Engel, virologists; and Dr. Dickenson, an expert in spinal and supraspinal pathways of visceral and somatosensory pain. We will pursue the following specific aims: Aim 1: Map the murine thoracic afferent pathways responsible for visceral pain evoked by the activation of capsaicin-sensitive (TRPV1+) DRG nociceptors. Aim 2: Map the contribution of peptidergic vs. non-peptidergic nociceptors to thoracic afferent pathways responsible for visceral pain evoked by the activation of capsaicin-sensitive (TRPV1+) DRG nociceptors. Aim 3: Map murine TRPV1-negative thoracic afferent pathways. Translational aim 4: Map TRPV1+ and Tac1+ esophageal afferents in rat and human tissue. We expect to identify and map the central pathways of specific DRG afferent subsets innervating the esophagus and lung. Furthermore, we expect the identified pathways connected to pain centers that are unique to esophageal (vs. lung) afferent innervation will be candidates for specifically mediating visceral pain. Our studies in mouse, rat and human tissue will increase our understanding of visceral pain.